The importance of new methods to regulate immune responses is underscored by the difficulty of generating effective vaccines for diseases like HIV, tuberculosis, or cancer. To exploit the full potential of the immune system, the antigen features that lead ultimately to tolerance or immunity must be defined. To expedite the generation of antigens that elicit desired immune responses, a fundamental knowledge is needed of how antigen processing cells (APCs) and the downstream responses that result are influenced by antigen structure. Chemical synthesis provides access to well-defined antigens that vary in valency, elasticity, the identity and number of immune receptors they target, and the proteases that can process them. The four Specific Aims have been devised to leverage synthetic methods from chemistry and chemical biology to generate tailored synthetic antigens to dissect critical steps in immune system function. In the previous grant period, we developed strategies to assemble chemically defined epitopes that target receptors on B cells or the lectins on dendritic cells. We demonstrated that by displaying specific epitopes on a polymer backbone, we could devise antigens that bind the B cell receptor to activate or tolerize B cells. We shall leverage the synthetic methods and building blocks that resulted from those investigations to synthesize chemically defined antigens to pinpoint attributes critical for immunity. A key element of our approach is the use of state-of-art polymer chemistry to vary antigen features, such that the requisite properties can be identified. In Aim 1, we shall use defined antigens to address how valency influences antigen signaling and trafficking in B cells and dendritic cells (DCs) but also how antigen valency influences the ability of these APCs to activate T cells. In Aim 2, we shall synthesize nanoparticles to test the hypothesis that increases in antigen stiffness will augment B cell and dendritic cell activation, facilitate antigen uptake and processing, and therefore promote immunity. In Aim 3, we shall produce antigens to exploit the vacuolar pathway to elicit cross presentation and thereby augment immunity by activating cytotoxic T cell responses. In Aim 4, we shall use the blueprints that emerge from Aims 1-3 to generate antigens that induce potent in vivo immune responses. We anticipate that progress on the proposed Aims will yield new strategies to recruit the immune system to treat human disease.